Intracellular signaling by the insulin receptor kinase

描述（由申请人提供）：本研究计划的总体目标是识别和表征胰岛素信号转导通路，导致直接参与含glut4囊泡的细胞内运输、质膜系结、对接和融合的蛋白质成分的功能调节。大量证据表明，质膜脂筏微域为胰岛素的生物作用提供了一个重要的空间限制区。这些微结构域自组织成大的玫瑰花状结构，高度富含小窝蛋白，作为质膜皮质肌动蛋白（Cav-actin）组装的支架或平台。虽然动态肌动蛋白重塑在GLUT4易位过程中起着重要作用，但其机制尚不清楚。我们的初步数据表明，肌动蛋白组装蛋白的特定子集（β 1整合素受体亚基、talin和I型磷脂酰肌醇-4-磷酸5激酶）与这些Cav-Actin结构域共定位，而不是其他。此外，GLUT4囊泡融合蛋白（SNARE）也定位于这些结构域，并似乎在功能上与F-actin相互作用。为了进一步研究脂肪细胞脂筏微结构域在介导胰岛素信号传导中的功能作用，我们提出了四个具体目标：1。我们将通过siRNA培养和体内同源重组来降低a1整合素受体亚基、talin和PI4P5激酶的表达。在这些实验中，我们将根据肌动蛋白的组装和胰岛素特异性信号过程的获取来评估这些蛋白质在体内和体外脂肪细胞分化过程中的作用。我们将评估几种SNARE蛋白在GLUT4囊泡对接与融合中的作用，对肌动蛋白组装的影响以及对脂筏微结构域定位的需求。3. 我们将重建膜囊融合，并确定这些调节蛋白对体外融合反应的影响。将确定直接激酶磷酸化和磷酸化缺陷突变体的影响。为了建立质膜融合实验，我们将评估这些重组膜囊泡与脂肪细胞质膜片之间的融合反应。4. 我们将使用FRAP来评估在各种条件下分离的质膜片和完整细胞中的扩散速率，包括脂筏、皮质肌动蛋白和微管的破坏和/或稳定。在互补的方法中，我们还将利用新开发的光激活GFP结构体（PA-GFP）来检测非脂质筏和富含小泡蛋白的质膜微域的扩散。

DESCRIPTION (provided by applicant): The overall goals of this research proposal are to identify and characterize the insulin signal transduction pathways leading to functional regulation of the protein components directly involved in the intracellular trafficking, plasma membrane tethering, docking and fusion of GLUT4-containing vesicles. Substantial evidence has indicated that plasma membrane lipid raft microdomains provide an important spatially restricted compartment necessary for insulin biological action. These microdomains are self-organized into large rosette-like structures highly enriched in caveolin that serve as scaffolding or platform for the assembly of plasma membrane cortical actin (Cav-actin). Although it is well-established that dynamic actin remodeling plays an essential role in the GLUT4 translocation process the mechanism(s) responsible are completely unknown. Our preliminary data indicates that a certain subset of actin assembly proteins (beta1 integrin receptor subunit, talin and the type I phosphatidylinositol-4-phosphate 5-kinase) but not others, are co-localized to these Cav-Actin domains. In addition, GLUT4 vesicle fusion proteins (SNARE) have also been localized to these domains and appear to functionally interact with F-actin. To further examine the functional role of adipocyte lipid raft microdomains in mediating insulin signaling, we propose four specific aims, 1. We will reduce expression of the a1 integrin receptor subunit, talin and PI4P5 kinase by the use of siRNA in culture and by homologous recombination in vivo. In these experiments, we will assess the role of these proteins during the adipocyte differentiation process both in vivo and in vitro in terms of the assembly of actin and acquisition of insulin specific signaling processes. 2 We will assess the role of several SNARE proteins in GLUT4 vesicle docking versus fusion, effects on actin assembly and requirement for lipid raft microdomain localization. 3. We will reconstitute membrane vesicle fusion and determine the effect of these regulatory proteins on the in vitro fusion reaction. The effect of direct kinase phosphorylation and phosphorylation deficient mutants will be determined. As efforts to establish a plasma membrane fusion assay, we will assess the fusion reaction between these reconstituted membrane vesicles with adipocytes plasma membrane sheets. 4. We will use FRAP to assess the diffusion rates in isolated plasma membrane sheets and intact cells under a variety of conditions including disruption and/or stabilization of lipid rafts, cortical actin and microtubules. In a complimentary approach, we will also take advantage of the newly developed photoactivable GFP construct (PA-GFP) to examine diffusion from non-lipid raft and caveolin-enriched plasma membrane microdomains.